The metabolism of inositolphospholipids is believed to be an essential part of the receptor-mediated signal transduction pathways in response to various hormones and growth factors [see, e.g., Berridge, M. J., et al., Nature, 312:315-321 (1984); Nishizuka, Y., Science, 225: 1365-1370 (1984)].
In this signaling pathway, two intracellular second messengers, inositol 1,4,5-trisphosphate and diacylglycerol are generated through the hydrolysis of phosphatidyl 4,5-bisphosphate by phospholipase C. Inositol 1,4,5-trisphosphate releases Ca.sup.2+ from intracellular Ca.sup.2+ stores leading to the activation of Ca.sup.2+ /calmodulin-dependent kinase; diacylglycerol activates protein kinase C. Following breakdown, phosphatidylinositol 4,5-bisphosphate is rapidly resynthesized by stepwise phosphorylation of phosphatidylinositol by phosphatidylinositol 4-kinase and phosphatidylinositol-4-phosphate kinase. These 2 kinases appear to play important roles in the production of second messengers (see, e.g., Duell, T. F., U.S. Pat. No. 5,001,064 (1991); Shibasaki, F., et al., J. Biol. Chem., 266 (13): 8108-8114 (1991).
More recently, the existence of another phosphatidylinositol kinase has been identified and associated with certain activated tyrosine kinases [Courtneidge, S. A., et al., Cell, 50:1031-1037 (1987); Kaplan, D. R., et al., Cell, 50: 1021-1029 (1987)]. This kinase, identified as phosphatidylinositol 3-kinase has been found to phosphorylate the 3-position of the inositol ring of phosphatidylinositol (PI) to form phosphatidylinositol 3-phosphate (PI-3P) [Whitman, D., et al., Nature, 332:664-646 (1988).
In addition to PI, this enzyme also can phosphorylate phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate to produce phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PIP.sub.3), respectively [Auger, K. R., et al., Cell, 57:167-175 (1989)].
PI 3-kinase physically associates with tyrosine kinases such as pp60.sup.v-src, polyoma middle T/pp60.sup.c-src, platelet-derived growth factor receptor, colony stimulation factor-1 receptor, and insulin receptor (see, e.g., Shibasaki supra), suggesting it has important, but yet undefined roles in signal transduction, mitogenesis, cell transformation, and other cellular events involving protein tyrosine kinases that associate with and activate PI 3-kinase. PI 3-kinase activity also has been identified in association with G-protein receptors in neutrophils and platelets in neutrophils [Traynor-Kaplan, A. E., et al., Nature, 334:353-356 (1988); and Mitchell, C. A., et al., Proc. Nat. Acad. Sci., 87:9396-9400 (1990)]. However, activation of PI 3-kinase in the neutrophil occurs independently of tyrosine phosphorylation [Vlahos, C. J., et al., FEBS Letters, 309(3):242-248 (1992)].
PI 3-kinase exists as a tightly associated heterodimer of an 85 kDa regulatory subunit and an 110 kDa catalytic subunit, and is found in cellular complexes with almost all ligand-activated growth factor receptors and oncogene protein tyrosine kinases [Cantley, L. C., et Cell, 64:281-302 (1991)]. The 85 kDa regulatory subunit apparently acts as an adaptor protein which allows the 110 kDa catalytic subunit of PI 3-kinase to interact with growth factor receptors and tyrosine phosphorylated proteins [Margolis, C., Cell Growth Differ., 3:73-80 (1992)].
Although PI 3-kinase appears to be an important enzyme in signal transduction, with particular implications relative to mitogenesis and the malignant transformation of cells, only a limited number of compounds have been identified as having inhibitory activity against PI 3-kinase [see, e.g., Matter, W. F., et al., Biochem. Biophys, Res. Commun., 186: 624-631 (1992)]. Contrary to the selective PI 3-kinase activity of the compounds used in the methods of the present invention, the bioflavinoid compounds used by Matter, et al., particularly quercetin and certain analogs thereof, inhibit PI 3-kinase and other kinases such as protein kinase C and PI 4-kinase (Matter, et al., supra).
Thus, the present invention provides a method for inhibiting phosphatidylinositol 3-kinase in a lysed or whole cell with wortmannin or one of certain wortmannin analogs.
The present invention also provides a method for inhibiting phosphatidylinositol 3-kinase in mammals, particularly humans, using wortmannin or one of certain analogs thereof.
Furthermore, the present invention provides a method for treating phosphatidylinositol 3-kinase-dependent conditions, particularly neoplasms, in mammals.